Dissertations / Theses on the topic 'Thraustochytrids'

Docosahexaenoic acid (DHA; 22:6 n-3), a polyunsaturated fatty acid (PUFA), is linked to various health benefits in humans including cognitive and visual development of infants and reduced risk of cancer, cardiovascular diseases and mental illnesses of adults. Fish

oil, with an annual production of about 600,000 tons is at present the major source of DHA. However, the production of fish oils is expected to become inadequate for supplying an expanding market within few years. Thraustochytrids are marine heterotrophic producers of PUFA-rich triacylglycerols which represent an alternative source of DHA.

The focus of this thesis has been split between a basic study of the osmolyte system of traustochytrids and work towards an understanding of their growth kinetics, effects of nutrient depletion, and lipid and DHA accumulation. Three new osmotolerant thraustochytrid isolates (T65, T66, and T67) and the previously known Schizochytrium sp. strain S8 (ATCC 20889) were assigned to the genus Aurantiochytrium based on 18S ribosomal DNA phylogeny, morphology and PUFA profiles (approximately 80% DHA). Strains T66 and S8 displayed a nearly linear increase in cellular content of endogenously synthesized (-)-proto-quercitol and glycine betaine with increasing osmotic strength. This represented the first demonstration of accumulation of principal compatible solutes in thraustochytrids. A less osmotolerant isolate (Thraustochytriidae sp. strain T29), which was closely phylogenetically related to Thraustochytrium aureum (ATCC 34304) did not accumulate glycine betaine or (-)-proto-quercitol, illustrating a variation in osmolyte systems and osmotolerance levels among thraustochytrids.

To study the effects of nutrient limitations, Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate and glycerol containing medium with various medium limitations. N and P starvation and O₂ limitation initiated lipid accumulation. N starvation alone or in combination with O₂ limitation yielded the highest lipid contents obtained in this study, i.e., 54 to 63% of cell dry weight with a corresponding cell density of 90 to 100 g l^-1 dry biomass. The DHA-content of N starved cells was 29% of total fatty acids, while O₂ limitation increased the DHA-content up to 52%. Simultaneously, O₂ limitation abolished accumulation of monounsaturated fatty acids. We inferred that the biological explanation is that O₂ limitation hindered activity of the O₂-dependent desaturase(s) responsible for production of monounsaturated fatty acids, and favored the O₂-independent PUFA synthase. The highest DHA-productivity observed was 93 mg l^-1 h^-l, obtained during sequential N starvation and O₂ limitation. This

productivity approaches the highest values previously reported for thraustochytrids, and indicates that T66 may become a candidate organism for a future large-scale microbial PUFA production process.

Omega-3 fatty acids, in particular docosahexaenoic acid (DHA), have been extensively studied for many decades for their health benefitting properties in pre-natal development, cardiovascular and Alzheimer diseases and enhancing the inflammatory immune response system. DHA has also been shown to be essential for the optimal development of fish, and therefore is an important ingredient in fish feed. However, due to fish oil and fish meal supplies currently facing many challenges (e.g. heavy metal contamination, environmental impacts, etc.), the demand for alternative sources of omega-3 fatty acids (FA) is predicted to rise in the near future. To meet these challenges, oleaginous microorganisms that produce omega-3 FAs have been explored as a potential new resource, with a particular emphasis on the thraustochytrid group. In this study, ten new strains of thraustochytrids, that were originally isolated from Scottish marine waters, were investigated for their biotechnological potential. The first phase of the project identified the new strains as a novel genus of thraustochytrid, for which the name Caledonichytrium matryoshkum gen. nov., sp. nov., is proposed. The description was based on a polyphasic analysis that employed phylogenetic analysis, biochemical signatures (PUFA and carotenoid profiles) and morphological and life cycle assessment studies. After identification, the strains were screened for their potential as DHA single-cell oil producers. The strains were assessed in two media types and at two time points of growth phase. With a view to their industrial application, a mathematical study was also included to seek opportunity for recycling by-product oil as biodiesel. The results showed that one of the strains, OL5TA, produced the highest relative level of DHA (63% of total FA) than any other strain reported to date in a screening study. However, the low final biomass concentrations reached (< 1 g L-1), and the low total lipid content measured (< 7% of dry cell weight, DCW), were considered major hurdles to overcome for industrial application. To address these issues, the optimisation of the culture conditions was carried out in the following stage. The results showed no consumption of glucose at 0.1% or 2% concentration, suggesting the inability of the strain to assimilate glucose. This may have hindered competitive biomass concentrations compared to that by other strains reported in the literature. To remediate this inability, a preliminary study was conducted to determine carbon source utilisation and carotenoid production to seek other routes for medium optimisation and biotechnological potential. The study concluded by identifying a potential route of exploitation for galactose and long carbon chain as sole carbon sources.

Thraustochytrids are well-reported microorganisms with an outstanding ability to produce and accumulate polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). In the present study, a lab-scale optimization on medium and fermentation parameters, was carried out with Aurantiochytrium sp. 0043 AA aiming at high cell biomass, lipids and DHA yields. Medium optimization was performed based on the literature, focused on the screening of different C and N sources, on the concentration of each source as well as on the ratio and feeding strategies. In addition, other fermentation parameters were optimized, such as initial inoculum conditions (volume, age and stage), different oxygen transfer rates and pH. Later on, the growth performances of Aurantiochytrium sp. 0050 AA and 0051 AA, were compared to strain 0043 AA. The respective cryopreservation processes weres completed and the banks validated, with the exception of the master cell bank (MCB1) of strain 0051 AA. Overall, lab-scale optimization of strain 0043 AA allowed to increase biomass yield by 2fold and improve lipid and DHA contents from 22.78% and 1.25% to 31.14% and 29.66%, respectively. Furthermore, with strain 0051 AA, a maximum dry weight (DW) of 80.15 g/L was obtained, with concomitant lipid and DHA contents of 60.34% and 38.81%, respectively. Although several conditions were tested in the 5 L bench-top reactor, cultivation was not successful due to the shear-sensitive cells of this species and further trials on fermentation parameters and reactor design are required to enable the scale-up.

碩士
國立中山大學
海洋生物科技暨資源學系研究所
107
Thraustochytrids is a kind of heterotrophic microalgae. The ratio of lipid to cell dry weight can reach 50%. It is considered to have the potential to develop the biofuel. In some species, even high valued compounds such as carotenoids are produced. This study selected Aurantiochytrium sp. AP45 as the optimal condition for the production of astaxanthin and life cycle assessment. During the screening stage of the medium, the results showed that the medium with high nitrogen content favored the accumulation of DHA. At the same carbon source concentration and the C/N ratio, the medium with low nitrogen content was more favorable for the accumulation of astaxanthin. Fermentation culture results showed that Aurantiochytrium sp. AP45 is not suitable for the cultivation of waste cooking oil as a carbon source. In addition, there is a significant difference in the accumulation of astaxanthin with or without LED blue light at Fed-batch cultivation. Aurantiochytrium sp. AP45 without LED blue light, the accumulation of astaxanthin per unit algae is 7.27 g/g, and the total yield of astaxanthin is 62.17 g/L. If it is with LED blue light during fermentation, the accumulation of astaxanthin per unit algae can reach 28.20 g/g, and the total output of astaxanthin is 172.06 g/L, which is about 4 times higher. The life cycle assessment of carbon footprint and environmental impact was performed on the results of the astaxanthin cultured in Aurantiochytrium sp. AP45. The functional unit was 1 g astaxanthin. The cultivation stage is a great emission source of carbon footprint and environmental impact. The carbon footprint of the cultivation stage is about 2,180 kgCO2e/g, mainly from the electricity required for LED illumination. The acetone used in the extraction stage is a great impact source at Carcinogens category . Most of the existing research on micro-algae put a lot of effort on algae biofuels. However, there are very few studies on health foods and cosmetics. The life cycle assessment of health foods for heterotrophic microalgae is almost none. Therefore, the present investigation contributes to the life cycle assessment study of the astaxanthin production in heterotrophic algae.

碩士
國立臺灣師範大學
生物研究所
87
Thraustochytrids are non-photosynthetic marine protists, belonging to chromists. They display an obligate sodium requirement, so they are widely distributing in the ocean, estuary, inland salt lakes and the mangroves. The thalli of thraustochytrids are similar to the monocentric chytrids. Thrausto-chytrids have biflagellate zoospores and non-flagellate aplanospores. They are saprophytic on algae and phanerogamic detritus and parasitic on invertebrate. In the asexual reproduction, the thallus includes the sporangium and the ectoplasmic net. So far, no sexual reproduction have been documented in any thraustochytrids. Ththraustochytrids are isolated from the soil and water samples collected in the north coast of Taiwan by using pine pollen grains (Pinus taiwanensis). All isolates were successively transferred by streaking on the KMV agar medium containing antibiotics until the cultures were purified. Then transferred the thraustochytrids on the KMV agar medium and in pollen seawater. In such culture conditions, observation and discription of their morphological characteristics were made. Totally, three genera and nine species are successfully examined and identified namely: five species of Thraustochytrium : T. motivum、T. antarcticum 、T. proliferum 、T. kinnei、T. aggregatum；two species of Ulkenia：U. visurgensis 、U. radiata；two species of Shizochytrium： S. octosporum 、S. mangrovei. Among them, T. antarcticum、T. kinnei、U. visurgensis、U. radiata、 S. octosporum and S. mangroveiare are described as new records to Taiwan.

碩士
國立臺灣師範大學
生物研究所
88
Abstract Thraustochytrids are non- photosynthetic marine protests, belonging to chromists. They display an obligate sodium requirement, so they are widely distributing in the ocean, estuary, inland salt lakes and the mangroves. The thalli of thraustrids are similar to the monocentric chytrids. Thraustochytrids have biflagellate zoospores and non-flagellate aplanospores. They are saprophytic on algae and phanerogamic detritus and parasitic on invertebrate. In the asexual reproduction, the thallus includes the sporangium and ectoplasmic net. So far, no sexual reproduction have been documented in any thraustochytrids. Thraustochytrids are isolated from the soil and water samples collected in the north coast of Taiwain by using pine pollen grains(Pinus taiwainensis). All isolates were successively transferred by streaking on the KMV agar medium containing antibiotics until the cultures were purified. Then transferred the thraustochytrids on the KMV agar medium and in pollen seawater. In such culture conditions, observation and discription of their morphological characteristics were made. Totally, three genera and nine species are successfully examined and identified namely : five species of Thraustochytrium : T. motivum 、T. antarcticum 、T. proliferum 、T. kinnei 、T. aggregatum；two species of Ulkenia : U. visurgensis 、 U. radiata ；two species of Schizochytrium ：S. octosporum S. mangrovei. Among them, T. antarctium、T. kinnei 、U. visurgensis、U. radiata、S. octosporum、S. mangrove are described as new records to Taiwain.

Dissertação de mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016
Microalgae are a promising source of biofuels and other valuable chemicals. The reduced economic feasibility of microalgal cultures is due to low cell density and slow growth rate of these cultures. Thus, it is necessary to develop sustainable processes that will be able to increase the productivity, maximize the production yield and reduce production costs. To achieve this goals, it is necessary to improve the understanding of the behavior of microalgal cultures. A robust method was developed for the growth of Thraustochytrids in saline waste medium using glycerol as carbon source. The first study case was the heterotrophic cultivation of Schizochytrium limacinum in order to achieve the highest growth and DHA content possible using alternative raw materials. In the second study case, the temperature profiles of Japanochytrium sp. were analyzed to identify the optimal temperature for growth and PUFAs production. The first case study, Schizochytrium limacinum was grown in saline waste medium using glycerol as carbon source. After 216 hours of fermentation the values obtained were the following: biomass concentration of 40.4 g.L-1, DHA content of 48.5% and a DHA productivity of 424 mg.L-1.d-1. The results obtained proved that Schizochytrium limacinum. is able to grow and produce high levels of DHA using saline waste medium and glycerol as carbon source. Besides, it was made the economic balance of the used media for biomass and DHA production. In standard medium to produce 1 kg of biomass and 1 kg of DHA will cost 105 € and 1.235 €, respectively. While in saline waste medium to produce 1 kg of biomass and 1 kg of DHA will cost 17.16 € and 180.7 €, respectively. The second case study, Japanochytrium sp. was cultivated at different temperatures of 15, 20, 25 and 30 ˚ C. The highest value of biomass, 22 g.L-1, was obtained at 25 ˚C whereas the lowest value of biomass, 11 g.L-1, was obtained at 15 ˚C. For 20 ˚C and 30 ˚C the biomass concentration was 19 g.L-1 and 16 g.L-1, respectively. These results are in agreement with the literature, where the optimal temperature to the growth of thraustochytrids range between 22-28 ˚C. Glycerol is the major byproduct of the biodiesel industry and since it is expensive to purify, biodiesel producers must seek alternative methods for its disposal. Hence using glycerol as a carbon source for fermentation is an alternative use for this product.
Com o decorrer do tempo as microalgas têm vindo a tornar-se uma fonte promissora no ramo dos biocombustíveis e industrias relacionadas. A baixa viabilidade económica no cultivo de microalgas deve-se a uma fraca densidade celular e baixa taxa de crescimento. Deste modo, é necessário desenvolver processos sustentáveis capazes de aumentar a produtividade, maximizar o rendimento e reduzir os custos de produção. Para atingir estes objetivos é necessário aprofundar conhecimentos para a compreensão do comportamento em culturas de microalgas. Neste trabalho, foi desenvolvido um meio de cultura para o crescimento de Thraustochytrids em meio salino, proveniente do efluente de uma fábrica de lacticínios e usando glicerol como fonte de carbono. O primeiro caso de estudo foi o cultivo heterótrófico de Schyzochytrium limacinum de modo a atingir uma elevada taxa de crescimento desta microalga bem como o conteúdo de ácido docosahexaenoico (ADH) associado usando matérias-primas alternativas. O segundo caso de estudo teve como objectivo identificar os perfis de temperatura da microalga Japanochytrium sp. de modo a identificar a temperatura ideal para o seu crescimento e para a produção de ácidos gordos polinsaturados. A microalga Schizochytrium limacinum foi cultivada em meio salino usando glicerol como fonte de carbono. Após 216 horas de fermentação obtiveram-se os seguintes resultados: concentração de biomassa de 40,4 g.L-1, conteúdo em ADH de 48,5% e a produtividade de ADH atingiu 424 mg.L-1.d-1. Os resultados obtidos provaram que a microalga em questão tem uma boa capacidade de crescimento aliado a uma produção elevada de ADH. Foi feito um balanço económico entre o meio salino standard e o meio salino do efluente para a produção de biomassa e ADH. Em meio standard para produzir 1 Kg de biomassa e 1 Kg de ADH custa 105 € e 1.235€, respectivamente. No segundo caso de estudo, o Japanochytrium sp. foi cultivado a diferentes temperaturas: 15, 20, 25 e 30 ˚C. O valor mais elevado de biomassa, 22 g.L-1, foi obtido a 25 ˚C e o valor mais baixo de biomassa, 11 g.L-1 foi obtido a 15 ˚C. Para 20 ˚C e 30 ˚C a concentração de biomassa obtida foi, 19 g.L-1 e 16g.L-1, respectivamente. Os resultados obtidos neste estudo estão de acordo com a literatura, onde a temperatura ótima de crescimento para o crescimento de thraustochytrids varia entre 22-28 ˚C. O glicerol é o principal subproduto na indústria do biodiesel e uma vez que os custos de purificação para uso na indústria farmacêutica e alimentar são demasiado elevados, os produtores devem procurar métodos alternativos para a sua utilização. Provou-se que utilizando o glicerol como fonte de carbono para fermentações é uma alternativa bastante atrativa para o uso deste produto.

碩士
國立臺灣海洋大學
海洋生物研究所
101
Thraustochytrids are unicellular, eukaryotic marine protists and belong to the Kingdom Chromista. Sodium is an obligate requirement for growth of thraustochytrids. They are widely distributed in the ocean, estuary, inland salt lakes and mangroves. They possess: (1) sagenogenetosome, (2) multilamellate cell wall, and (3) biflagellate zoospores. In the environment, thraustochytrids can produce extracellular enzymes that decompose leafy tissues for nutrition. No sexual reproduction has been reported for thraustochytrids. In Taiwan, high level of heavy metals has been detected in many mangroves and it has been proven to affect morphology and physiological of thraustochytrids. In this study, we investigated the effects of various concentration of Cu (II) ion on growth and production of zoospores of thraustochytrids. Fifteen Schizochytrium isolates from four mangroves, including Tamsui in New Taipei City, Xinfeng in Hsinchu, Chunan in Miaoli, and Beimen in Taiwan. Results showed that the growth of Schizochytrium spp. ( IMB176、177、178、179、180、182、183、184、185、186、187、188 ) was inhibited at 32 ppm Cu (II), while two other isolates of Schizochytrium spp. IMB174 and, IMB181 were inhibited at 16 ppm Cu (II). Production of zoospores under the influence of Cu (II) by different Schizochytrium islolates varied. Schizochytrium spp. IMB174、IMB182、IMB183、IMB185 only produced zoospores at concentration below 2 ppm Cu (II). Schizochytrium spp. IMB176、IMB179、IMB181、IMB184、IMB186、IMB188 were capable of producing zoospores up to 128 ppm Cu (II). Schizochytrium spp. IMB175、IMB177、IMB178、IMB180、IMB187 could produce zoospores up to 256 ppm Cu (II). Results suggested that there were different responses on growth and sporulation by the fifteen Schizochytrium isolates under various Cu (II) concentrations. The results of this study will contribute to the understanding of heavy metal pollutants on the ecology of this important group of organisms.

碩士
國立中山大學
海洋生物科技暨資源學系研究所
107
Thraustochytrids are marine unicellular protist. They are similar to the common heterotrophic bacteria, which relies on the decomposition of organic matter from other organisms. They show absolute demand for sodium, widely distributed in mangroves, sea areas or estuaries. They can produce some high-priced compounds, such as astaxanthin. In this study, the feasibility of algae for production of astaxanthin was to screen the algae strain with the highest astaxanthin content, and the astaxanthin production was increased by using different culture conditions and waste cooking oil. The results showed that 25 strains of Thraustochytrids were isolated from mangroves. Among them, 14 strains were known as Thraustochytrids, 1 strain was yeast, and the remaining 10 strains had not been determined. The strain YA7.1, who was selected from Kaohsiung Yong-an mangrove and showed the highest astaxanthin production, was used in the following experiments. In the experiment of optimizing the medium, the carbon source was glucose or waste cooking oil, and the nitrogen source was tryptone and yeast extract. The best astaxanthin yields for glucose and waste cooking oil were 197 μg/L and 70 μg/L, respectively using 125 ml flask culture system. Then, by using the fermenter culture, the best astaxanthin productions were 320 μg/L and 308 μg/L for glucose and waste cooking oil, respectively. After the blue LED illumination using glucose as the carbon source, the production of astaxanthin was twice that of the unilluminated control, but the use of waste cooking oil as the carbon source, the production of astaxanthin was 10 times lower. Based on the above results, it is feasible to use waste cooking oil as a carbon source to produce astaxanthin, but the increase in the production of astaxanthin by blue LED illumination was only workable using glucose as the carbon source.

碩士
國立臺灣海洋大學
海洋生物研究所
97
Abstract The objectives of this thesis are: (1) to investigate the growth and morphological effects of increasing heavy metal stress on three strains of thraustochytrids isolated from fallen mangrove leaves collected in Kaohsiung Erjen river estuary, Tainan Yan-Tian ecological park mangrove and Miaoli Chu-Nan mangrove, caused by three heavy metals commonly exist in Taiwan’s metal-polluted river: copper, zinc and lead, and (2) to trace the fate of the selected heavy metal ions using atomic absorption spectrophotometer (AAS). Experiment on growth indicated that EC60 of Schizochytrium sp. EJ56 and Schizochytrium sp.CN75 was 64 ppm Zn2+, while EC60 of Schizochytrium sp.YT90 and Schizochytrium sp.CN75 was 128 ppm Cu2+ and EC60 of all strains was 512 ppm Pb2+. These results suggest that the toxicity of heavy metals on growth of thraustochytrids is in the order: Zn2+> Cu2+> Pb2+. Schizochytrium sp.CN75 was most tolerant to Cu2+ and Zn2+ while Schizochytrium sp.YT90 grew best in the presence of lead. Among all the isolates tested, Schizochytrium sp.CN75 was the most tolerant to the selected heavy metal ions. These suggest that thraustochytrids isolated from the river near the industry park (Schizochytrium sp.EJ56) may be more sensitive when facing to heavy metal stress. Observation of morphological changes on thraustochytrids under elevated heavy metal pressures under SEM (scanning electron microscope), heavy metals led to the formation of holes on cell wall, shrinkadge of cells and deformation of cell. At highest metal concentrations, cell lysis was observed and intracellular content leaked out. Under differential interference contrast microscope, perfectly round cells were observed in control and deformed cells were seen at the highest metal concentration. We detected heavy metal reduction by treating quantitative concentrations of heavy metals before and after thraustochytrids growing. The appearance of copper, zinc and lead reduction indicated that thraustochytrids can reduce in vitro heavy metals during their growth and the reduction rate was: Pb2+ > Zn2+ > Cu2+. The most significant reduction range were between 1 ppm and 256 ppm. The mechanism of heavy metal reduction by thraustochytrids remains unknow. We look forward follow-up studies to find out if the missing metals were adsorpted or absorpted by thraustochytrids, or both pathways contribute to the results. The results in the current study clearly shown that in the presence of high concentrations of heavy metal ions, growth of thraustochytrids would be retarded, eventually causing death. More effort should be done on the effects of heavy metal ions on the ecology of thraustochytrids, an important decomposer in marine environment.

碩士
大葉大學
生物產業科技學系碩士在職專班
96
In the last few years , everyone started paying attention to personal health, and disease prevention due to the improvement of the society. Docosahexaenoic acid (DHA) has physiological effects in heart and circulatory system, inflammatory and dermatitis disease. The study was investigated on batch production of DHA by Thraustochytrium sp. using 20 L fermentor. During the batch cultivation, temperature and pH were controlled at 25 ℃ and 6.5. Agitation and airflow were adjusted to keep the dissolved oxygen above 30 ％ of Saturation. Samples were taken for measurements of biomass, lipid and DHA contents. The medium contained per liter:20.0 g glucose, 1.0 g ammonium sulfate. The results of experiment show that after 30 hrs, the nitrogenous source was exhausted, but there was still 11.54 g/L of carbonic source in the broth. However, the biomass and lipid were keeping raising. After 33 hrs, the biomass and lipid increased greatly. Until glucose was exhausted, biomass and lipid was decreasing. Under no carbonic source, microbe would consume the lipid which made by itself so that lipid was just decreasing. At 37 hrs, biomass was 3.48 g/L and lipid was 1.58 g/L. It was close to the stationary phase of microbial growth and lipid. At this time, docosahexaenoic acid was 251.07 mg/L. The lipid was 48 ﹪of biomass and the doc- osahexaenoic acid was 7.61 ﹪of biomass, respectively. Key Words：Thraustochytrium sp. docosahexaenoic acid (DHA)

碩士
大葉大學
生物產業科技學系碩士在職專班
96
Docosahexaenoic acid is a Polyunsaturated fatty acid that cannot be synthesized in human cell, and therefore must be supplied in the diet .It can help to lower levels of serum cholesterol and triglycerides and to prevent hypertension ; it also has anti- inflammation function. Thraustochytrium sp. was screened for the production of Docosahexaenoic acid.in this study, response surface methodology was used to study the growth of Thraustochytrium sp.,the production of lipid and to develop a numerical model.The method of one-factor-at-a-time was used to investigate the effects of cultivating conditions, including the carbon and nitrogen sources,concentrations of glucose and ammonium chloride, initial pH ,and temperature on the microbial growth and lipid production by Thraustochytrium sp. Then, the central composite design was used to obtain optimal production of lipid. The results by one-factor-at-a-time method showed that Thraustochytrium sp. was best cultivated in a medium with 40 g/L glucose and 1.0 g/L ammonium chloride,under 25℃and pH 6.0. Based on the resuts by one-factor-at-a-time,a central composite design of three factors, each with two levels,was designed,and 8 factorial,6 compensate and 2 central experiments were conducted. The ranges for the ammonium chloride concentration and the pH were the same in both cultures of Thraustochytrium sp. The concentration of glucose at the central point was 40g/L . The experiments were carried our and results were analyzed with statistical software,STATISTICA. The optimal cultivating condition for Thraustochytrium sp. was as follows: glucose 40.52 g/L,ammonium chloide1.07 g/L, and pH6.19, and the Lipid production 227.69 mg/L(95% of the expected result) , R2＝0.9261. Under the optimal conditions bactch cultivation of Thraustochytrium sp.were conducted in a 5L fermentor. Cultivate Thraustochytrium sp. at 25℃ for 99hr, the biomass was 2.3 g/L , it is 2.7 times more than in a shaker with the optimal conditions; the lipid production is 943 mg/L , it is 4.35 times more than in a shaker with the optimal conditions .

The aim of this research was to isolate and characterize novel strains of marine protists with potential to commercially produce PUFAs. Twelve trips were made visiting 10 different locations in the Canadian Maritime Provinces. Sixty-nine strains were isolated and screened for biomass and fatty acid production. Those meeting specific criteria were selected for further investigation including characterization by 18S rDNA sequencing. Isolate ONC-KTB-56 produced the greatest amount of biomass (1 807 mg L-1) and fatty acids (24.6% dry weight). Of the total fatty acids, ARA, EPA and DHA comprised 0.89, 1.22 and 4.7 percent, respectively. Isolate ONC-KTB-14 produced 1 704 mg L-1 dry biomass with 5.4 percent fatty acids including 1.44, 1.35 and 37.5 percent, ARA, EPA and DHA, respectively. Through optimization of culture conditions biomass, fatty acid content and the proportions of specific fatty acids can be increased. With such optimization, there is potential for isolates ONC-KTB-14 and ONC-KTB-56 to be grown at a commercial scale for production of PUFAs.

碩士
國立成功大學
化學工程學系
104
Docosahexaenoic acid (DHA), the omega-3 polyunsaturated fatty acid (ω-3 PUFA) helps in preventing cardiovascular diseases and Alzheimer's disease. The World Health Organization (WHO) has recommended a daily intake of 250 mg ω-3 PUFA, especially for infants, elderly and pregnant women. The major industrial source of DHA is fish oil, yet there are concerns regarding the sustainability of fishes, contamination, and its complex fatty acid profile. Thus, Thraustochytrium sp., a group of heterotrophic marine protists, has earned much attention recently to serve as an alternative to fish oil, due to their high DHA content and productivity. This objective of this study is comprised of four main parts. A heterotrophic microalga with a high cellular DHA content was isolated and examined for its potential to serve as a promising alternative source for DHA. The first part of this research was to find a suitable cryoprotective additive (CPA) and storage method for the prolonged preservation of this strain. The results showed that when the strain was preserved in 12.5% glycerol and stored in Liquid Nitrogen (-196oC), highest survival rate of 85.24% was obtained after one-year of freezing. In the second part, the selection of flask closures and types and optimal cultivation conditions (salinity, temperature and pH) for cell growth and DHA accumulation were conducted with the strain cultivated in shake flasks and in serum bottles. A combination of SILICOSEN® type cap and un-baffled flasks yielded the highest biomass and DHA productivity of 1170 and 64 mg/L/d respectively. Next, while the strain was cultivated in serum bottles with aeration and agitation, the highest biomass productivity (1960 mg/L/d) and DHA productivity (339 mg/L/d) was obtained under the optimal culturing conditions of 3% sea salt, 25oC, pH 7.5, 15 g/L of glucose, 5 g/L of peptone, and 5 g/L of yeast extract. The third part aimed to improve the DHA productivity of the microalga by optimizing the operating conditions in bioreactors. The effects of different agitation rate (0, 50, 100 and 150 rpm) and aeration rate (increased from 0.4 to 1.4 vvm) on the microalgal biomass productivity and DHA productivity were studied in 5-L stirred-tank fermentor. The results showed that when the strain was cultivated at an agitation rate of 100 rpm and an aeration rate of 1.2 vvm (resulting in a KLa level of 27±1.1 hr-1), the highest biomass productivity (4.59 g/L/d) and DHA productivity (379 mg/L/d) was achieved. The effect of different concentration of glucose (15, 30, 40, 60, 75 and 90 g/L) on DHA production was also studied, and the highest biomass productivity (5580 mg/L/d) and DHA productivity (457 mg/L/d) was obtained with 60 g/L glucose. Moreover, because the decreasing cellular DHA content was observed when increasing agitation rate due to the shear sensitivity of this strain, the effect of pneumatic and mechanical agitation under the same KLa was studied with two different types of fermentors (stirred-tank and air-lift). The results showed that when this strain was cultivated in air-lift fermentor, better biomass concentration (27.57 g/L), cellular DHA content (13.1 %) and DHA productivity (610 mg/L/d) were achieved. Finally, in the fourth part of this thesis, to avoid the corrosive effects of chloride ions on the fermentor, sodium sulfate was used to replace the artificial sea salts added in the original medium. The results obtained from batch cultures (200 ml working volume) showed that biomass productivity improved from 1.19 to 1.44 g/L/d, while the DHA productivity also slightly increased from 89 to 96 mg/L/d when sodium sulfate replaced sodium chloride. The outcome of this work could provide useful information and knowledge on attaining high cell density fermentation and improving DHA production with Thraustochytrium sp. BM2 and demonstrates the feasibility of replacing sea salts with sodium sulfate without any considerable loss in DHA productivity.